Methods and compositions to detect nucleic acids in a biological sample

ABSTRACT

Kits, reaction mixtures and methods for separating a target nucleic acid from a sample by using at least one hairpin capture probe oligonucleotide that has the structure 5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Y each comprise nucleic acid sequences that can form a double stranded stem portion, one of X or Y is a capture sequence that is a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and a′ b′ c′ comprises a target-complementary sequence flanked by X and Y to thereby form a loop portion of the hairpin, thus forming a capture hybrid that is separated from other sample components before the target nucleic acid is released from the capture support and hybridized to a detection probe that hybridizes specifically to the same sequence that is at least partially hybridized by the a′ b′ c′ portion of the capture probe, thus forming a detectable detection hybrid to indicate the presence of the target nucleic acid in the sample.

RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 11/173,915, filed Jun. 29, 2005 and claims priority to provisionalapplication No. 60/585,421, filed Jul. 1, 2004, both of which areincorporated by reference herein.

FIELD OF THE INVENTION

The invention is related to the field of nucleic acid assays, andparticularly to detection of a specific target nucleic acid present in asample, such as a small RNA or DNA sequence present in a biologicalsample.

BACKGROUND

Detection of nucleic acids in a sample is useful in diagnostic,therapeutic, forensic, agricultural, food science applications and otherareas. Methods of nucleic acid detection include those that use physicalseparation and detection of a nucleic acid, such as by capturing thenucleic acid in or on a matrix or support and detecting the capturednucleic acids by using a means to visualize the nucleic acid, such as adye or intercalating agent, or by hybridizing a detectable probe to thenucleic acid. Known methods for separating and detecting nucleic acidsuse electrophoretic separation of nucleic acids by size, e.g. by using agel or other chromatographic matrix, followed by staining or attaching aprobe to the separated nucleic acids to produce a signal to indicate thepresence of the nucleic acid in the sample. Some methods indirectlydetect nucleic acids by producing a product made from using a targetnucleic acid as a template and detecting the product, e.g., detecting anRNA transcript made from a DNA, or a translated protein made from an RNAtranscript. Other indirect methods detect a product made by an enzymaticreaction associated with the nucleic acid to be detected, e.g., anenzyme-linked probe hybridized to the target nucleic acid which producesa detectable response when the enzyme's substrate is provided. Somemethods of nucleic acid detection rely on amplifying a nucleic acidsequence to produce a larger quantity of nucleic acid that is detected.Examples of amplification methods include producing many copies of acloned sequence and in vitro amplification procedures that use enzymaticsynthesis of multiple copies of a nucleic acid sequence.

Many of the techniques for detecting nucleic acids require the presenceof a relatively large amount or proportion of the target nucleic acid inthe sample, while other techniques use nucleic acid amplification toincrease the amount or proportion of the nucleic acid to be detectedfrom a smaller amount of the target nucleic acid in a sample. Enrichmentof some or all of the nucleic acid present in a sample may facilitatedetection of the nucleic acid of interest. Many known procedures fornucleic acid enrichment and detection are laborious, time-consuming, orrequire use of equipment or hazardous chemicals (e.g., chaotropes,mutagens, or radioactive compounds) that make such proceduresundesirable for many applications, such as for rapid screening of manysamples, point-of-care diagnostics, or detection at a site outside of alaboratory. Thus, there remains a need for a method that providesrelatively simple procedures and sufficient sensitivity and/orspecificity to detect a nucleic acid of interest.

The physical nature or relative abundance of some nucleic acids mayimpede their detection in a sample. For example, small RNA (about 17-27nt), such as microRNA (miRNA), small or short interfering RNA (siRNA),short hairpin RNA (shRNA), and small nuclear RNA (snRNA) are difficultto separate from other sample components and/or to detect by using knownmethods. Small RNA are often relatively rare in a biological samplewhich contributes to the difficulty of their detection. Because smallRNA are important regulatory molecules that modulate or silence geneexpression via RNA interference (RNAi), they may be important diseasepreventive or therapeutic agents. Thus, there is a need for a methodthat rapidly detects the presence of small RNA in biological samples todetermine their presence, stability, therapeutic efficacy, or othercharacteristics in a biological sample without requiring extensiveprocessing or nucleic acid amplification. There is a further need todetect localized small RNA in a variety of biological samples to avoidconditions that lead to inadvertent suppression of non-targeted genefunctions by small RNA. Current methods for detecting small RNA or theireffects in biological samples are time consuming and laborious, e.g., insitu hybridization, nuclease protection assays, Northern blots to detectRNA, Western blots to detect proteins, immunoassays, and fluorescencedetection assays (PCT App. Nos. WO 0044914, Li et al., WO 05004794,Bumcrot et al.).

This application responds to the need for efficient nucleic aciddetection assays by disclosing methods and compositions useful for therapid detection of nucleic acids in samples, including small RNA inbiological samples.

SUMMARY OF THE INVENTION

An aspect of the invention is a method of detecting the presence of anucleic acid present in a sample which includes the steps of: providinga sample containing a target nucleic acid, mixing the sample with anucleic acid capture probe that forms under hybridizing conditions apartially double-stranded hairpin structure made up of an internaltarget-complementary sequence, flanked by a capture region, and aterminal region that binds to the capture region to form adouble-stranded stem portion of the hairpin structure in which thetarget-complementary region forms a substantially single-stranded loopportion, specifically hybridizing the target-complementary sequence ofthe capture probe to a target sequence in the target nucleic acid,binding the capture region to an immobilized probe attached to a capturesupport by binding together members of a specific binding pair, therebyforming a capture hybrid made up of the target nucleic acid, the captureprobe, and the immobilized probe attached to the capture support,separating the capture hybrid attached to the capture support fromsample components, releasing the target nucleic acid from the capturehybrid, then specifically hybridizing a detection probe to the targetnucleic to form a detection hybrid, and detecting a signal produced fromthe detection hybrid to indicate the presence of the target nucleic acidin the sample. In one embodiment, the capture region is located near the3′ end of the capture probe and the terminal region is located near the5′ end of the capture probe. In another embodiment, the capture regionis located near the 5′ end of the capture probe and the terminal regionis located near the 3′ end of the capture probe. In one embodiment, thestep of binding the capture region to the immobilized probe hybridizescomplementary sequences of the capture region and the immobilized probe.In another embodiment, binding the capture region to the immobilizedprobe binds together non-nucleic acid members of a specific bindingpair, such as a ligand and its receptor. In one embodiment, releasingthe target nucleic acid from the capture hybrid further releases thecapture probe from the immobilized probe. In one embodiment, thedetecting step uses a detection probe that hybridizes specifically to atarget sequence that is the same target sequence that hybridizes to thetarget-complementary sequence of the capture probe. In anotherembodiment, the detection probe hybridizes specifically to a targetsequence that differs from or overlaps the target sequence thathybridizes to the target-complementary sequence of the capture probe. Ina preferred embodiment, the detecting step detects a signal is producedin a homogeneous reaction.

Another aspect of the invention is a method of detecting the presence ofa target nucleic acid present in a sample that includes the steps of:providing a sample containing a target nucleic acid, mixing the samplewith a capture probe that is at least a partially double-strandedstructure made up of a first strand and a second strand of nucleic acid,wherein the first strand includes a target-complementary region and acapture region, and the second strand contains a sequence complementaryto a sequence of the first strand, specifically hybridizing thetarget-complementary region of the capture probe to a target sequence inthe target nucleic acid, binding the capture region to an immobilizedprobe attached to a capture support, thereby forming a capture hybridmade up of the target nucleic acid, the first strand of the captureprobe, and the immobilized probe attached to the capture support,separating the capture hybrid attached to the capture support from othersample components, releasing the target nucleic acid from the capturehybrid, then specifically hybridizing a detection probe to the targetnucleic acid to form a detection hybrid, and detecting a signal producedfrom the detection hybrid, thereby indicating the presence of the targetnucleic acid in the sample. In one embodiment, the first strand containsa 5′ capture region covalently linked to a 3′ target-complementaryregion, and the second strand contains a 3′ sequence complementary tothe capture region of the first strand, thereby forming a partiallydouble-stranded structure when the capture region of the first strandhybridizes to the complementary 3′ sequence of the second strand. Inanother embodiment, the first strand contains a 5′ target-complementaryregion covalently linked to a 3′ capture region, and the second strandcontains a 5′ sequence complementary to the 3′ capture region of thefirst strand, thereby forming a partially double-stranded structure whenthe capture region of the first strand hybridizes to the complementary5′ sequence of the second strand.

Another aspect of the invention is a nucleic acid capture probe thatforms at least a partially double-stranded structure under hybridizingconditions and includes a target-complementary sequence and a captureregion that binds to an immobilized probe by using members of a specificbinding pair. In one embodiment, the partially double-stranded structureis a hairpin structure made up of a contiguous linear sequence thatincludes an internal target-complementary sequence, flanked by thecapture region and a terminal region that binds to the capture region toform a double-stranded stem portion of the hairpin structure and thetarget-complementary region forms a substantially single-stranded loopportion of the hairpin structure. It will be appreciated that thecapture region may be a 5′ region and the terminal region is a 3′ regionof the contiguous linear sequence, or alternatively, the capture regionmay be a 5′ region and the terminal region is a 3′ region of thecontiguous linear sequence that forms a hairpin structure. In anothercapture probe embodiment, the structure is made of up of a first strandthat includes the target-complementary region and the capture region,and a separate second strand that includes a sequence complementary to asequence of the first strand such that hybridization of thecomplementary sequences of the first strand and second strand produce atleast a partially double-stranded structure. It will be appreciated thatthe first strand may have a 5′ target-complementary region and a 3′capture region, or alternatively, a 3′ target-complementary region and a5′ capture region, and that the complementary sequence of the separatesecond strand may be complementary to either the target-complementaryregion or the capture region of the first strand. In a preferredembodiment, the complementary sequence of the second strand iscomplementary to the capture region of the first strand. Anotherembodiment is a kit that includes the capture probe that forms apartially double-stranded hairpin structure and a detection probe thathybridizes specifically to a target nucleic acid that contains asequence that hybridizes to the target-complementary sequence of thehairpin structure capture probe. In one embodiment of the kit, thedetection probe hybridizes specifically to the same target nucleic acidsequence that hybridizes to the target-complementary sequence of thecapture probe. In another embodiment, the kit further includes animmobilized probe attached to a capture support, in which theimmobilized probe includes a member of a specific binding pair thatbinds specifically to the capture probe. In one kit, the specificbinding pair members are complementary sequences that hybridize thecapture probe to the immobilized probe under hybridizing conditions.Another kit embodiment includes the capture probe made of up of a firststrand that includes the target-complementary region and the captureregion and a separate second strand that includes a sequencecomplementary to a sequence of the first strand, and a detection probethat hybridizes specifically to a target nucleic acid that contains asequence that hybridizes to the target-complementary sequence of thatcapture probe.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an embodiment showing capture and detection of atarget nucleic acid by mixing a target nucleic acid (shown as thesequence a b c d) with a single-stranded capture probe to form a capturehybrid made up of the target nucleic acid hybridized totarget-complementary region (shown as the sequence a′ b′ c′) of thecapture probe and an immobilized probe (shown as poly-T attached to acapture support) hybridized to another portion of the capture probe(shown as poly-A), followed by release of the target nucleic acid andformation of a detection hybrid made up of a detection probe (shown asthe sequence a′ b′ c′ d′) hybridized to the target nucleic acid, wherethe detection hybrid produces a detectable signal (shown by the blackstar icon) to indicate the presence of the target.

FIG. 2 illustrates an embodiment showing capture and detection of atarget nucleic acid by mixing target nucleic acid (shown as the sequenced c b a) with a hairpin capture probe which has complementary sequencesat its 5′ and 3′ ends (shown as poly-T and poly-A regions) flanking atarget complementary region (shown as the sequence a′ b′ c′ d′) to forma capture hybrid made up of the target nucleic acid hybridized totarget-complementary region of the opened capture probe, and a portionof a capture probe (poly-A region) hybridized to a complementaryimmobilized probe (shown as poly-T attached to a capture support),followed by releasing the target into solution where it forms adetection hybrid made up of a detection probe (shown by the sequence d′c′ b′ a′) hybridized to the target nucleic acid to produce a detectablesignal (shown by the black star icon) to indicate the presence of thetarget.

FIG. 3 illustrates an embodiment showing capture and detection of atarget nucleic acid that mixes the target nucleic acid (shown as thesequence a b c d e) with a completely or partially double-strandedcapture probe that contains complementary sequences on the two strands(shown as poly-A and poly-T sequences) and one target-complementaryregion (shown as the sequence a′ b′ c′ on the poly-T containing strand),to form a capture hybrid made up of the target nucleic acid hybridizedto the target-complementary region of the capture probe strand, andanother portion of the capture probe strand (poly-T) hybridized to acomplementary immobilized probe (shown as poly-A attached to a capturesupport), followed by releasing the target nucleic acid into solution toform a detection hybrid made up of a detection probe (shown by thesequence a′ b′ c′ d′ e′) hybridized to the target nucleic acid toproduce a detectable signal (shown by the black star icon) to indicatethe presence of the target.

DETAILED DESCRIPTION

This invention is useful for detection of a target nucleic acid ofinterest present in a sample. The methods use relatively few and easilyperformed steps to isolate and/or concentrate the target nucleic acidfrom other sample components and to detect the target nucleic acid. Themethods include a capture step in which the target nucleic acid iscaptured by using one or more capture probes to form of a capture hybridthat is linked to a capture support, which together are separated fromother sample components. Then, the target nucleic acid in the capturehybrid is released from the capture support and the target nucleic acidis detected by using a detection probe to form a detection hybrid thatcauses production of a detectable signal. Although any specific bindingpair may be used to link the components of the capture hybrid and thedetection hybrid, preferred embodiments use nucleic acid hybridizationto form the capture and detection hybrids. In a preferred embodiment,the detection step is performed in a homogeneous reaction assay in whichunbound detection probe does not interfere with detection of a signalresulting from the bound detection probe in the detection hybrid. Thiscontributes to the simplicity and efficiency of the system becauseunattached detection probe does not have to be removed from the mixturebefore detection of the signal from the detection hybrid.

These methods are useful particularly for detecting small target nucleicacids that may be present at dilute concentrations in a sample, e.g.,small nucleic acids excreted in urine or present in a cellular or tissueextract. These methods are also useful for assaying many samples,preferably simultaneously or in rapid succession, such as in anautomated high through-put system because the capture and detectionsteps can be performed in a single reaction chamber per sample.

To better understand the various embodiments of the invention, some ofthe terms used in the description of the invention are more fullydescribed below.

By “nucleic acid” is meant a polydeoxyribonucleotide (DNA or an analogthereof) or polyribonucleotide (RNA or an analog thereof) made up of atleast two, and preferably ten or more bases linked by a backbonestructure. In DNA, the common bases are adenine (A), guanine (G),thymine (T) and cytosine (C), whereas in RNA, the common bases are A, G,C and uracil (U, in place of T), although nucleic acids may include baseanalogs (e.g., inosine) and abasic positions (i.e., a phosphodiesterbackbone that lacks a nucleotide at one or more positions, U.S. Pat. No.5,585,481). Nucleic acids include polynucleotides or polymers andoligonucleotides or oligomers of DNA and RNA, which may besingle-stranded (ss), double-stranded (ds), or triple-stranded.Oligomers generally refer to nucleic acids that comprise 1,000 or fewernucleotides, and often comprise two to about 100 nucleotides, whereaspolymers generally refer to nucleic acids that comprise 1,000 or morenucleotides, including nucleic acid structures comprised of manythousands of nucleotides, such as plasmids, cosmids, genes, chromosomes,genomes and the like which are well known in the art. Also included inthe term are “locked nucleic acids” (LNA), a nucleic acid analogue thatcontains one or more LNA nucleotide monomers with a bicyclic furanoseunit locked in an RNA mimicking sugar conformation, to enhancehybridization affinity toward complementary ssRNA, or complementaryssDNA or dsDNA (Vester B. and Wengel J., 2004, Biochemistry.43(42):13233-41).

A nucleic acid “backbone” refers to groups or linkages known in the art(Eschenmoser, 1999, Science 284:2118-2124), such as, e.g.,sugar-phosphodiester linkages, 2′-O-methyl linkages, guanidine linkersin DNA (“DNG”), S-methylthiourea linkers, methylphosphonate linkages,phosphoramidite linkages, amide backbone modifications as in polyamideor peptide nucleic acids (PNA), phosphorothioate linkages, phosphonicester nucleic acid linkages, pyranosyl oligonucleotide linkages,bicyclo- and tricyclo-nucleic acid linkages, formacetal and3′-thioformacetal linkages, morpholino linkages, or other modificationsof the natural phosphodiester internucleoside bond, or combinations ofsuch linkages in a single backbone (Majlessi et al., 1998, Nucl. AcidsRes. 26(9):2224-2229; Dempcy et al., 1995, Proc. Natl. Acad. Sci. USA92:6097-6101; Browne et al., 1995, Proc. Natl. Acad. Sci. USA92:7051-7055; Arya & Bruice, 1998, J. Am. Chem. Soc. 120:6619-6620;Reynolds et al., 1996, Nucl. Acids Res. 24(22):4584-4591; Gryaznov &Chen, 1994, Am. Chem. Soc. 116:3143-3144; Chaturvedi at al., 1996, Nucl.Acids Res. 24(12):2318-2323; Hyrup & Nielsen, 1996, Bioorg. & Med. Chem.4:5-23; Hydig-Hielsen et al., PCT App. No. WO 95/32305; Mesmaeker etal., Syn. Lett., November 1997:1287-1290; Peyman et al., 1996, Angew.Chem. Int. Ed. Engl. 35(22):2636-2638; Aerschot et al., 1995, Angew.Chem. Int. Ed. Engl. 34(12):1338-1339; Koshkin et al., 1998, J. Am.Chem. Soc. 120:13252-13253; Steffens & Leumann, 1997, J. Am. Chem. Soc.119:11548-11549; Jones et al., 1993, J. Org. Chem. 58:2983-2991;Summerton & Weller, 1997, Antisense & Nucl. Acid Drug Dev. 7:187-195;Stirchak et al., 1989, Nucl. Acids Res. 17(15):6129-6141). A nucleicacid backbone may include a mixture of linkages in the same nucleic acid(e.g., sugar-phosphodiester and 2′-O-methyl linkages) or may have all ofone type of linkages (e.g., all 2′-O-methyl or all amide modificationlinkages in an oligomer).

A “target” or “target sequence” or “target nucleic acid” refers to asequence of nucleotide bases present in a nucleic acid, or portion of anucleic acid, to which another sequence binds, e.g., by using standardcomplementary base pairing. For example, the target sequence may be arelatively small part of a larger nucleic acid, such as a specificsubsequence contained in a gene or messenger RNA (mRNA). Those skilledin the art will appreciate that a target nucleic acid may exist indifferent forms, i.e., single-stranded, double-stranded,triple-stranded, or mixtures thereof, such as in a partiallydouble-stranded hairpin structure or partially double-stranded duplexstructure, and will further appreciate that a target sequence may bepresent on any strand (+ or −) of the structure. For simplicity, atarget nucleic acid may be described as all or part of a single strand,but this is not meant to limit the meaning of a target to one or aparticular nucleic acid strand. It is well known in the art that amulti-stranded nucleic acid is readily converted to its single-strandcomponents by using standard methods, such as by heating a nucleic acidabove its melting temperature (Tm) and/or by using chemical denaturants.

By “complementary” or “complementarity of” nucleic acids is meant that anucleotide sequence in one strand of nucleic acid, due to orientation ofthe functional groups, will hydrogen bond to another sequence on anopposing nucleic acid strand. The complementary bases typically are, inDNA, A with T and, C with G, and, in RNA, C with G, and U with A.“Substantially complementary” means that a sequence in one strand is notcompletely and/or perfectly complementary to a sequence in an opposingstrand, but that sufficient bonding occurs between bases on the twostrands to form a stable hybrid complex in set of hybridizationconditions (e.g., salt concentration and temperature). Such conditionscan be predicted by using the sequences and standard mathematicalcalculations known to those skilled in the art to predict the Tm ofhybridized strands, or by empirical determination of Tm by using routinemethods. Tm refers to the temperature at which a population ofhybridization complexes formed between two nucleic acid strands are 50%denatured. At a temperature below the Tm, formation of a hybridizationcomplex is favored, whereas at a temperature above the Tm, melting orseparation of the strands in the hybridization complex is favored. Tmmay be estimated for a nucleic acid having a known G+C content in anaqueous 1 M NaCl solution by using, e.g., Tm=81.5+0.41 (% G+C), althoughother Tm computations are known in the art which take into accountnucleic acid structural characteristics.

“Hybridization condition” refers to the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions and hydrogen bonding to produce ahybridization complex. Such conditions include the chemical componentsand their concentrations (e.g., salts, chelating agents, formamide) ofan aqueous or organic solution containing the nucleic acids, and thetemperature of the mixture. Other well known factors, such as the lengthof incubation time or reaction chamber dimensions may contribute to theenvironment (e.g., Sambrook at al., Molecular Cloning, A LaboratoryManual, 2^(nd) ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).

A “label” refers to a molecular moiety that is detectable or produces adetectable response or signal directly or indirectly, e.g., bycatalyzing a reaction that produces a detectable signal. Labels includeluminescent moieties (such as fluorescent, bioluminescent, orchemiluminescent compounds), radioisotopes, members of specific bindingpairs (e.g., biotin and avidin), enzyme or enzyme substrate, reactivegroups, or chromophores, such as a dye or particle that results indetectable color.

A “detection probe” is an oligomer or polymer that binds specifically toa target sequence and which binding results, directly or indirectly, ina detectable signal to indicate the presence of the target sequence. Adetection probe need not be labeled to produce a detectable signal,e.g., an electrical impulse resulting from binding the probe to itstarget sequence may be the detectable signal. A “labeled probe” is aprobe that contains or is linked, directly or indirectly, to a label.Methods of making and/or using labeled probes are well known in the art(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.,Chapt. 10; U.S. Pat. Nos. 6,361,945, Becker et al.; 5,658,737, Nelson etal.; 5,656,207, Woodhead et al.; 5,547,842, Hogan et al.; 5,283,174,Arnold et al.; 4,581,333, Kourilsky et al.; 5,731,148, Becker et al.).For example, detection probes may include a non-nucleotide linker and achemiluminescent label attached to the linker (U.S. Pat. Nos. 5,185,439,5,585,481 and 5,639,604, Arnold et al.).

“Homogeneous detectable label” refers to a label whose presence can bedetected in a homogeneous manner depending on whether the label is boundor unbound to a target. A homogeneous detectable label can be detectedin a “homogeneous reaction” without physically separating unbound formsof the label from the mixture before the detection step. It will beappreciated that a homogeneous reaction may occur in solution or on asupport such as a microarray, biochip, or gene chip. Preferredhomogeneous detectable labels and conditions for their detection havebeen described previously in detail (U.S. Pat. Nos. 5,283,174, Arnold etal.; 5,656,207, Woodhead et al.; 5,658,737, Nelson et al.).

An “immobilized probe” provides a means for joining a capture hybridcontaining a target nucleic acid to a capture support. A preferredimmobilized probe is a nucleic acid oligomer or polymer joined to asupport which binds, directly or indirectly, to a target nucleic acid tofacilitate separation of the bound target nucleic acid from unboundmaterial, such as other sample components. In a preferred embodiment,the target nucleic acid is indirectly bound to the immobilized probe viaa capture probe. Any of a variety of materials may be used as a capturesupport, e.g., matrices or particles made of nitrocellulose, nylon,glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene,and magnetically attractable materials. Monodisperse magnetic spheresare a preferred embodiment of a capture support because they arerelatively uniform in size and readily retrieved from solution byapplying a magnetic force to the reaction container, preferably in anautomated system. An immobilized probe may be linked directly to thecapture support, e.g., by using any of a variety of covalent linkages,chelation, or ionic interaction, or may be linked indirectly via one ormore linkers joined to the support.

A “capture probe” provides a means for joining a target nucleic acid andan immobilized probe, preferably by hybridization of complementarysequences. A capture probe includes a target-complementary region ofsequence and a means for attaching the capture probe, or a hybridizationcomplex that includes the capture probe, to an immobilized probe. Suchattaching means may be a region of sequence that is complementary to asequence of an immobilized probe, or a member of another specificbinding pair (e.g., biotin and avidin or streptavidin). In a preferredembodiment, a capture probe includes is a nucleic acid homopolymer(e.g., poly-A or poly-T) that is covalently attached to thetarget-complementary region of the capture probe and that hybridizesunder appropriate conditions to a complementary homopolymer of theimmobilized probe (e.g., poly-T or poly-A, respectively) as previouslydescribed (U.S. Pat. No. 6,110,678 to Weisburg et al.).

A “sample” or “biological sample” refers to any composition or mixturein which a target nucleic acid of interest may be present, including butnot limited to plant or animal materials, waste materials, materials forforensic analysis, environmental samples, and the like. A biologicalsample includes any tissue, cell, or extract derived from a living ordead organism which may contain a target nucleic acid, e.g., peripheralblood, bone marrow, plasma, serum, biopsy tissue including lymph nodes,respiratory tissue or exudates, gastrointestinal tissue, urine, feces,semen, or other body fluids.

“Separating” or “isolating” or “purifying” refers to removing one ormore components from a complex mixture, such as a sample. Samplecomponents may include target and non-target nucleic acids, and othermaterials such as salts, acids, bases, detergents, proteins,carbohydrates, lipids and other organic or inorganic materials.Preferably, a separating, isolating or purifying step removes at least70%, preferably at least 90%, and more preferably about 95% of thetarget nucleic acids from other sample components. A separating,isolating or purifying step may optionally include additional washingsteps to remove non-target sample components.

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating atarget nucleic acid from a capture probe, and/or a capture probe from animmobilized probe. Release of the target nucleic acid strand separatesthe target from other components of a capture hybrid and makes thetarget available for binding to a detection probe. Other components ofthe capture hybrid may remain bound, e.g., the capture probe strand tothe immobilized probe on a capture support, without affecting targetdetection. Release of one or more capture hybrid components may beaccomplished by changing one or more conditions to promote dissociationof components (e.g., heating above Tm, changing salt concentrations,adding denaturants or competitive binding moieties to the mixture), orby using other well known methods such as strand displacement.

“Consisting essentially of” is used to mean that additionalcomponent(s), composition(s) or method step(s) that do not materiallychange the basic and novel characteristics of the present invention maybe included in the compositions, kits or methods of the presentinvention. Such characteristics include the ability of atarget-complementary region of an oligomer to bind or hybridizespecifically to a target nucleic acid in a sample, the ability of acapture hybrid to be separated from other sample components, and theability of a detection probe to hybridize to the target nucleic acid andprovide a detectable signal to indicate the presence of the target in asample. These characteristics include the structural features of thecapture probes as described herein, which do not rely on any particularsequence as illustrated in FIGS. 1 to 3. It will be appreciated by thoseskilled in the art that a variety of conditions may be used to create acapture hybrid and a detection hybrid, and further that a variety ofdevices or systems may be used to accomplish the method steps of theinvention. Any component(s), composition(s), or method step(s) that havea material effect on the basic and novel characteristics of the methodsof the present invention, as exemplified by the embodiments describedherein, would fall outside of this term.

Examples of capture probes are oligomers of DNA, RNA and/or analogsthereof that are comprised of sequences of at least 10 nucleotidescomplementary to a target nucleic acid. Preferred embodiments includeoligomers that contain a target-complementary region of about 20nucleotides that has 2′-O-methyl linkages or other modified structure toenhance binding. Embodiments of capture probes include oligomers thathave a target-complementary sequences of about 15 to 25 nucleotidescovalently attached to a homopolymer sequence at the 3′ and/or 5′regions of the capture probe. Some embodiments include a poly-dAsequence of about 15 to 30 nucleotides covalently attached to one end ofthe target-complementary region of the capture probe.

Some capture probe embodiments include an oligomer that contains a 5′region, a middle target-complementary region, and a 3′ region, that canbe diagrammed as: 5′ X.sub.n a′ b′ c′ Y.sub.n 3′, in which X.sub.nindicates sequence X that comprises n residues, a′ b′ c′ indicates thetarget-complementary sequence, and Y.sub.n indicates sequence Y thatcomprises n residues, in which the X.sub.n and Y.sub.n regions can forma double-stranded structure so that the entire capture probe forms ahairpin structure with the target-complementary sequence as the loop.Some embodiments include complementary homopolymeric sequences at the 5′X.sub.n and 3′ Y.sub.n regions that flank the target-complementaryregion of the linear capture probe so that under hybridizing conditionsit forms a partially double-stranded hairpin structure by intramolecularhybridization of the homopolymeric sequences. Examples of hairpincapture probes include a 5′ poly-dT region adjacent to thetarget-complementary region and a 3′ poly-dA region so that thetarget-complementary region forms the loop of the hairpin structure whenthe poly-dT and poly-dA regions are bound to each other, as illustratedin FIG. 2. In this embodiment, the target-complementary region remainssubstantially single-stranded in the hairpin structure. Those skilled inthe art will understand that any complementary sequences located in the5′ and 3′ regions may be used to flank the target-complementary regionin an oligomer that forms a hairpin capture probe structure.

Other capture probe embodiments include partially or completelydouble-stranded structures made up of two oligomer strands in which atleast a portion of each of the individual single strands iscomplementary to a portion of the opposing single strand. Suchembodiments can be diagramed as:

(first strand) 5′ X.sub.n a′ b′ c′ 3′ (second strand) 3′Y.sub.n a b c 5′in which X.sub.n indicates sequence X that comprises n residues, a′ b′c′ indicates the target-complementary sequence, a b c indicates anoptional sequence complementary to the target-complementary sequence,and Y.sub.n indicates sequence Y that comprises n residues, in which theX.sub.n and Y.sub.n regions can form a double-stranded structure. Forexample, a first single strand contains a 5′ poly-dT portion for X.sub.ncovalently linked to a 3′ target-complementary region, and a secondsingle strand contains a 3′ poly-dA portion for Y.sub.n covalentlylinked to another 5′ sequence region which may be complementary to aportion of the first strand, where the double-stranded structure is madeup of at least the hybridized poly-dT/poly-dA regions. Such a captureprobe is illustrated in FIG. 3.

Other embodiments of capture probes are single-stranded oligomers madeup of a 3′ or 5′ target-complementary region and a contiguous regionthat binds to an immobilized probe, which can be diagramed as:

5′ X.sub.n a′ b′ c′ 3′, or 5′ a′ b′ c′ X.sub.n 3′in which X.sub.n indicates sequence X that comprises n residues, and a′b′ c′ indicates the target-complementary sequence. One such embodimentis illustrated in FIG. 1.

Examples preferred embodiments of detection probes includeoligonucleotides of about 5 to 50 nucleotides in length having anattached label that is detected in a homogeneous reaction, e.g., onethat uses differential hydrolysis of a label on a bound or unboundprobe. Preferred embodiments of detection probes have a nucleotidesequence that is of the same sense as the target-complementary sequenceof the capture probe used in the assay. Other preferred embodiments ofdetection probes include those of the same nucleotide sequence as thetarget-complementary sequence of the capture probe. Preferred detectionprobes have an attached chemiluminescent marker, e.g., an acridiniumester (AE) compound (U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and5,656,744). In preferred embodiments, an acridinium ester label isattached to a central region of the probe near a region of A and T basepairs by using a non-nucleotide linker (U.S. Pat. Nos. 5,585,481 and5,656,744, Arnold, et al.) which restricts the amines of the nucleotidebases on both sides of the AE and provides a site for intercalation.Alternatively, an AE label may be attached to the 3′ or 5′ terminus ofthe detection probe which is used in conjunction with a second oligomerthat hybridizes adjacent to the detection probe on the target nucleicacid to restrict the effects of nearby amine contributed by the targetnucleic acid. Another embodiment attaches an AE label at or near thesite of a mismatch with a related non-target polynucleotide sequence, topermit discrimination between the related sequence and the targetsequence that may differ by only one nucleotide because the area of theduplex around the mismatch site is sufficiently destabilized to renderthe AE on the probe hybridized to the related non-target sequencesusceptible to hydrolysis degradation.

The methods of the present invention combine the steps of isolating atarget nucleic acid from a sample with detecting the target nucleic acidby using a detection probe to produce a detectable signal when thetarget nucleic acid is present in the sample. Compositions of theinvention include the structural capture probes, immobilized probes oncapture supports, and detection probes described herein, which may beincluded in kits with other reagents for performing the methods. Thefigures illustrate certain embodiments of the invention schematically.

FIG. 1 illustrates a method embodiment that uses a single-strandedcapture probe to capture a target nucleic acid from a sample, followedby detection of the target nucleic acid by forming a detection hybrid toproduce a detectable signal. The capture probe (shown as a sequence ofpolyA a′ b′ c′) and target nucleic acid (shown as a sequence of a b c d)are mixed in solution. Those skilled in the art will understand that adouble-stranded target nucleic acid may be treated using standardmethods to chemically or physically dissociate the strands and makesingle strands accessible for hybridization with the capture probe. Thecapture probe includes a target-complementary region (sequence a′ b′ c′)and another portion (polyA) that binds with an immobilized probe (shownas a poly-T attached to a capture support). Those skilled in the artwill recognize that any member of a specific binding pair, includingother nucleic acid sequences, may be substituted for the illustratedpoly-A region, so long as the specific binding pair member of thecapture probe binds to the immobilized probe which is or contains theother member of the specific binding pair.

The capture probe forms a capture hybrid by hybridizing thetarget-complementary region of the probe to the target nucleic acid andhybridizing or otherwise binding to an immobilized probe attached to acapture support. The immobilized probe, shown as a poly-T oligomerattached to the capture support, is complementary to the poly-A portionof the capture probe. The capture hybrid may be formed by simultaneouslybinding the immobilize probe, capture probe and target nucleic acid, ormay be formed in sequential steps. For example, sequential formation ofthe capture hybrid may first form a complex made up of the capture probeand the target nucleic acid, and then the complex attaches to the solidsupport by binding a portion of the capture probe to the immobilizedprobe. Another example of sequential formation first attaches thecapture probe to the immobilized probe and then binds the target nucleicacid to the capture probe at its target-complementary region. Followingformation of the capture hybrid, the capture hybrid is isolated fromother sample components by physically separating the capture supportusing any of a variety of known methods, e.g., centrifugation,filtration, magnetic attraction of a magnetic capture support. Tofurther facilitate isolation of the target nucleic acid from othersample components that adhere non-specifically to any portion of thecapture hybrid, the capture hybrid may be washed one or more times todilute and remove other sample components. Washing may be accomplishedby dissociating the capture hybrid into its individual components in anappropriate aqueous solution (e.g., 10 mM Tris, 1 mM EDTA) andappropriate conditions (e.g., temperature above the Tm of thecomponents) and then readjusting the conditions to permit reformation ofthe capture hybrid. For ease of handling and minimization of steps,washing preferably rinses the intact capture hybrid attached to thecapture support in a solution by using conditions that maintain thecapture hybrid.

Next, the target is released from the capture hybrid. For example, thecapture hybrid is released into its individual components to free thetarget nucleic acid into solution which makes it available to form adetectable complex with a detection probe. As illustrated, the captureprobe oligomer is released into solution but will not hybridize to thedetection probe because the capture and detection probe oligomers arestrands of the same sense. A detection probe (shown as a′ b′ c′ d′) isprovided in appropriate conditions to hybridize with the target nucleicacid (shown as a b c d), thus forming a detection hybrid. Because thereleased capture probe may compete with the detection probe forhybridization to the target nucleic acid, those skilled in the art willappreciate that the detection probe should be provided in excess orexhibit higher affinity (compared to the capture probe) for the targetnucleic acid by virtue of the detection. probe's length and/orstructural modifications (e.g., backbone). As illustrated the detectionprobe has a longer target-complementary sequence than the captureprobe's target-complementary sequence. The target nucleic acid isillustrated as approximately the same length as the detection probe, butthose skilled in the art will recognize that the detection probe may beshorter than the target nucleic acid and bind to a target region whichdiffers from the target region recognized by a capture probe.Optionally, one or more additional oligomers may bind to the targetnucleic acid in the detection step to facilitate binding the detectionprobe and/or producing a detectable signal. Such additional oligomersinclude, e.g., helpers, competitive probes for cross-reacting non-targetsequences, or an oligomer that brings another component used in signalproduction (e.g., enzyme, substrate, catalyst, or energy emitter) intoproximity with the detection probe (U.S. Pat. Nos. 5,030,557, Hogan etal.; 5,434,047, Arnold; and 5,928,862, Morrison). The detection stepuses conditions appropriate for production of a detectable signal (shownas a black star) from the detection hybrid, using methods well known inthe art. Detection of a signal from the detection hybrid indicates thepresence of the target nucleic acid in the sample.

FIG. 2 illustrates another method embodiment which uses similar steps tothose illustrated in FIG. 1, but uses a capture probe that forms ahairpin structure, which is partially double-stranded and contains asingle-stranded loop region that includes the target-complementaryregion. The capture probe includes complementary 5′ and 3′ sequences(shown as poly-T and poly-A), which form the double-stranded portion ofthe hairpin, flanking the target-complementary region (shown as a′ b′ c′d′), which forms the loop. In this embodiment, the 5′ and 3′ ends of thehairpin capture probe are separated (e.g., heating above the Tm todissociate the hydrogen bonds) to make a linear single-stranded captureprobe before hybridization with the target nucleic acid (shown assequence d c b a). A double-stranded target in the sample may also bemelted in a single melting step that linearizes the capture probe anddissociates that target nucleic acid strands before hybridization of thecapture probe and the target strand. Alternatively, hybridization of thetarget-complementary region with the target may occur (e.g., by stranddisplacement or strand invasion) without a melting step, thus separatingthe 5′ and 3′ ends of the capture probe. Excess capture probes that donot hybridize to the target would reform the hairpin structure byintramolecular hybridization to effectively prevent binding of thecapture probe to other components in the mixture, such as theimmobilized probe.

In this embodiment, the capture hybrid is made up of the target nucleicacid hybridized to the target-complementary region of the capture probeand one end of the capture probe hybridized to a complementaryimmobilized probe. As illustrated, a 3′ poly-A region of the captureprobe hybridizes to an immobilized probe of poly-T attached to thecapture support. Then, the solution phase is separated from the capturehybrids attached to the capture support and optional washing step(s) maybe used to further remove sample components, including excess hairpincapture probes unbound to the target. The method proceeds as describedabove for FIG. 1, by releasing the target from the capture hybrid orseparating the capture hybrid into its components, and then forming adetection hybrid made up of a detection probe (shown as sequence a′ b′c′ d′) hybridized specifically to the target nucleic acid. Asillustrated, the capture probe released from the capture hybrid reformsthe hairpin structure and will not hybridize to the immobilized probebecause of the preferred intramolecular hybridization or the detectionprobe because it is the same sense strand as the detection probe. Thisis an advantage of this embodiment because it prevents or minimizescompetition between the capture probe and the detection probe forbinding to the target. The detection hybrid made up of the detectionprobe and the target produces a detectable signal (shown as a blackstar) to indicate the presence of the target in the sample.

FIG. 3 illustrates a method embodiment in which the target nucleic acidis captured by using a completely or partially double-stranded captureprobe that contains complementary sequences on two strands (shown as a3′ poly-A region on one strand and a 5′ poly-T region on the otherstrand) and at least one target-complementary region (shown as a′ b′ c′on the poly-T containing strand). In this embodiment, only one strand ofthe capture probe hybridizes to the target nucleic acid (shown assequence a b c d e). It is important that the capture probe strand thatbinds to the target sequence also contains a specific binding partnermember that binds to the immobilized probe (shown as a poly-A strand onthe capture support). The partially double-stranded capture probe isusually dissociated before forming the capture hybrid although stranddisplacement caused by the target binding to the target-complementaryregion of the capture probe may separate the capture probe strands. FIG.3 illustrates a completely double-stranded embodiment in which the twostrands contain portions that are complementary to each other (shown aspoly-A on one strand, and poly-T on the other strand), and one strandcontains a target-complementary sequence whereas the other strandcontain a sequence complementary to the target-complementary sequence.Those skilled in the art will appreciate that the capture probe may bepartially double-stranded (e.g., substituting a polyA strand for thepolyA-a b c strand shown in FIG. 3). For completely and partiallydouble-stranded capture probes, the same assay steps are used,optionally starting with separation of the capture probe strands usingstandard methods to allow hybridization of the target-complementaryportion of one capture probe strand to the target nucleic acid. Becausethe two capture probe strands can rehybridize (e.g., via poly-A bindingto polyT) and interfere with the target-complementary sequence of thecapture probe strand binding to the target nucleic acid, those skilledin the art will appreciate that the capture probe may be synthesizedwith modifications to optimize hybridization to the target nucleic acid.

In this embodiment, the capture hybrid is made up of the target nucleicacid hybridized to the target-complementary region of one capture probestrand, and another portion of the same capture probe strand (shown aspoly-T) is bound to an immobilized probe (shown as poly-A) attached to acapture support. As described above, the capture hybrid attached to thesupport is separated from other sample components and, optionally,washed to remove sample components and capture probe strands unbound tothe capture hybrid. Capture probe strands that do not bind to the targetsequence can reform the partially or completely double-strandedstructure and be washed away, along with unbound single strands. Then,the target nucleic acid is released from the capture hybrid or thecapture hybrid is separated into its components, and the released targetis bound in solution by a detection probe (shown as sequence a′ b′ c′ d′e′) to form a detection hybrid that produces a signal (shown as a blackstar) which is detected to indicate the presence of the target nucleicacid in the sample. The released capture probe strand from the capturehybrid remains in solution as illustrated in FIG. 3, but does not bindthe detection probe because it is the same sense strand as the detectionprobe. Those skilled in the art will appreciate that the detection probeshould include structure that favors its binding to the target nucleicacid (e.g., increased sequence length and/or backbone modifications) tominimize competition between the detection probe and the releasedcapture probe strand for binding to the target.

A typical assay that uses a method described herein involves providing asample containing a nucleic acid of interest. Such a sample may be useddirectly in the assay or prepared by using any of a variety of methods,from simple dilution of a biological fluid with a lysing solution tomore complex methods that are well known in the art (e.g., Su et al., J.Mol. Diagn. 2004, 6:101-107; Sambrook, J. et al., 1989, MolecularCloning, A Laboratory Manual, 2^(nd) ed., pp. 7.37-7.57; and U.S. Pat.Nos. 5,374,522, 5,386,024, 5,786,208, 5,837,452, and 6,551,778).Typically, a sample containing a target nucleic acid is heated toinactivate enzymes in the sample and to make the nucleic acids in thesample single-stranded (e.g., 90-100.deg.C. for 2-10 min, then rapidlycooling to 0-5.deg.C.). To form a capture hybrid, the sample isincubated in appropriate hybridization conditions with a capture probe(e.g., any of the forms described above) and an immobilized probeattached to a capture support. An efficient method mixes thesecomponents together in a hybridization mixture and uses first conditionsto promote hybridization between the target-complementary region of acapture probe strand and the target nucleic acid, followed by secondconditions to promote binding of the capture probe:target complex to theimmobilized probe. For example, the first conditions may incubate themixture at a temperature below the Tm for the target-complementarysequence of the capture probe and the target sequence but above the Tmfor hybridization of sequences that bind the capture probe and theimmobilized probe, followed by incubating at a second temperature belowthe Tm for the capture probe binding to immobilized probe sequences(U.S. Pat. No. 6,110,678). In embodiments in which the capture hybrid isattached to the capture support by using members of a specific bindingpair that do not require nucleic acid hybridization (e.g., biotin andavidin or streptavidin), appropriate conditions for the selected bindingpair members are used. Following formation of the capture hybrids, thecapture hybrids attached to the capture support are separated physicallyfrom other sample components by using well known methods appropriate forthe support, e.g., removing a filter, membrane, or particles from thesolution phase by using filtration, centrifugation, gravity, magneticforce, and the like. When the capture support with attached capturehybrids have been separated from other sample components, optionalwashing steps may be included to further purify the captured targetnucleic acid, preferably performed while maintaining the capture hybridattached to the capture support. Then the target nucleic acid or allcomponents of the capture hybrid are released into solution to free thetarget for the detection step. Release of the target or capture hybridcomponents may be performed by any known method, such as, e.g., changingthe temperature or chemical composition of the mixture to promotedissociation of the capture hybrid into one or more of its nucleic acidcomponents. Typically, a simple heating step is performed to melt thetarget and capture probe strands, e.g., in an aqueous solution of lowionic strength, at 90-100.deg.C. for 5 min, followed by rapid cooling to0-5.deg.C. Other components of the capture hybrid may be released (e.g.,capture probe), but only the target nucleic acid must be made availableto bind to the detection probe. The soluble phase containing thereleased target nucleic acid may be separated from other components ofthe mixture (e.g., capture support and/or unbound capture probes) butthis is not critical because the capture probe strand is of the samesense as the detection probe and, therefore, will not interfere with thedetection probe binding to the target. As illustrated in FIG. 2, someembodiments further sequester the capture probe by reforming viaintramolecular hybridization the hairpin form of the capture probes. Thedetection step may be performed in soluble phase by adding a detectionprobe directly to the soluble phase containing the released targetnucleic acid and incubating the mixture in hybridization conditionssuitable for binding the detection probe and target sequences (e.g.,adding salts to the soluble phase to make a solution of suitable ionicstrength and incubating at 25-60.deg.C.). After the detection probebinds to the target nucleic acid to form the detection hybrid, a signalfrom the hybrid is detected to indicate the presence of the target inthe tested sample. Routine procedures may be used to remove unbounddetection probe before signal detection. In a preferred embodiment, thesignal from the detection hybrid is detected in a homogeneous reactionto avoid having to separate the unbound probes before signal detectionfrom the bound probes (e.g., as described in U.S. Pat. Nos. 5,283,174,5,639,604, 5,948,899, 5,658,737, 5,756,709, 5,827,656, and 5,840,873).Conditions suitable for producing and detecting a signal from the chosenlabel in the detection hybrid are well known to those of ordinary skillin the art.

The invention also includes kits containing components for performingthe methods for detecting target nucleic acids described herein.Preferred kits contain at least one detection probe specific for thetarget nucleic acid and a means for forming a capture hybrid containingthe target nucleic acid. Exemplary kits include a single-strandedcapture probe containing a target-complementary region and a means forbinding to an immobilized probe, with a detection probe specific for thetarget. Other exemplary kits include a capture probe containing atarget-complementary region flanked by two complementary regions thatform a hairpin structure under hybridization conditions, where one ofthe complementary regions serves as a means for binding the captureprobe to an immobilized probe, with a target-specific detection probe.Another exemplary kit includes a completely or partially double-strandedcapture probe containing in one strand a target-complementary region anda means for binding to an immobilized probe, with a target-specificdetection probe. Exemplary kits may further contain one or moreimmobilized probes attached to a capture support, where the immobilizedprobe is capable of binding to a portion of the capture probe(s) in thekit, such as by containing a complementary nucleotide sequence to aportion of the capture probe(s) or by containing a member of a specificbinding pair (e.g., avidin) that binds to its other binding pair memberon the capture probe (e.g., biotin). In preferred kits, the capturesupport is a magnetized particle, preferably a paramagnetic bead, withhomopolymeric oligomers (e.g., polyA, polyT, polyC, or polyG) attachedto it that are complementary to a homopolymeric portion of the captureprobe in the kit. Kit embodiments may also contain chemical compoundsused in forming the capture hybrid and/or detection hybrid, such assalts, buffers, chelating agents, and other inorganic or organiccompounds. Kit embodiments may contain chemical compounds used inreleasing the target nucleic acid from a capture hybrid, such as salts,buffers, chelating agents, denaturants, and other inorganic or organiccompounds. Kit embodiments may contain chemical compounds used in thedetection step, such as enzymes, substrates, acids or bases to adjust pHof a mixture, salts, buffers, chelating agents, and other inorganic ororganic compounds used in producing a detectable signal from a detectionhybrid. Kit embodiments may also contain chemicals for preparing samplesfor use in the invention methods which may include individual componentsor mixtures of lysing agents for disrupting tissue or cellular materialand preserving the integrity of nucleic acids. Such compositions includeenzymes, detergents, chaotropic agents, chelating agents, salts,buffering agents, and other inorganic or organic compounds. Kits mayinclude any combination of the capture probe, detection probe, andimmobilize probe components described above which are packaged incombination with each other, either as a mixture or in individualcontainers. It will be clear to skilled artisans that the inventionincludes many different kit configurations.

Aspects and embodiments of the present invention are illustrated in theExamples that follow. Methods and reagents for nucleic acid synthesis,hybridization, and detection of labels were used substantially asdescribed herein, although those skilled in the art will appreciate thatother routine methods and standard reagents may also be used to achieveequivalent results. Oligonucleotides were synthesized using standardphosphoramidite chemistry (Caruthers of al., 1987, Methods in Enzymol.,154: 287), purified using routine chromatographic methods (e.g., HPLC),and typically stored in a solution of 10 mM Tris, 1 mM EDTA (pH 7.5), atroom temperature to −80.deg.C. In the target capture steps illustratedin the examples, magnetic particles were used as the capture supportwhich were separated from the soluble phase by applying a magnetic fieldto the outside of the assay container, although those skilled in the artwill appreciate that other means of separation may be used. Thesupernatant containing soluble components was removed, and thehybridization complexes bound to the particles were washed (one to threetimes with a washing solution of sufficient ionic strength to maintainbonds binding the captured hybrid to the magnetic particles at thewashing temperature, usually about 25.deg.C.). Washing generally isperformed at room temperature by suspending the particles in the washingsolution, separating particles, and removing the supernatant, andrepeating those steps for each wash. For the detection step, thedetection probe was incubated with the released target nucleic acid inan aqueous solution containing appropriate salts and buffers at atemperature below the Tm predicted for the detection probe sequence andits target sequence, usually for 30-60 min. When an AE-labeled detectionprobe was used, a homogeneous detection step was performed which usesdifferential hydrolysis of the AE label on unbound probes compared toAE-labeled probes bound to the target (described in detail in U.S. Pat.No. 5,283,174). For example, hydrolysis was performed by adding aselection reagent that promotes hydrolysis of the AE label on unboundprobes (e.g., a basic solution), followed by adding a detection reagentthat catalyzes chemiluminescence from AE attached to bound probes (e.g.,H.sub.2O.sub.2), and the chemiluminescent signal (referred to asrelative light units or RLU) was detected on a luminometer (e.g.,LEADER® 450HC+, Gen-Probe Incorporated, San Diego, Calif.).

The following examples describe some preferred embodiments and reagentsused in these assays. The skilled artisan will appreciate that otherreagents and conditions may be substituted for those described herein toperform the method steps. For example, the reagents and conditions forproducing and detecting a signal will be selected by the skilled artisanbased on the chosen detection probe label. Those skilled in the art willunderstand that the invention methods may be performed using any chosentarget nucleic acid sequence that can hybridize to a complementarysequence, i.e., the method is not dependent on any particular probe ortarget sequences. One of ordinary skill in the art will be able toselect the target sequence and then design and synthesize theappropriate target-complementary sequence of any of the capture probeforms described herein, and a target-specific detection probe by usingroutine methods. That is, specific assays will rely on selection of atarget sequence and the appropriate target-complementary sequencescontained in the capture and detection probes that include thestructural characteristics described herein, and such selection can beperformed by one of ordinary skill in the art using standard procedures,followed by routine testing of the designed components to optimizedetection of the selected target by using the methods described herein.

EXAMPLE 1 Detection of Different Labeled Probes

To design detection probes, a target sequence of 23 nt was selected froma sequence common to genomic sequences of human Herpesvirus 5(Cytomegalovirus) strains (Dunn et al., 2003, Proc. Natl. Acad. Sci. USA100(24): 14223-14228; GenBank accession nos. AC146999, AC146851, andAY315197) and complementary to portions of fluorescent protein genes(GenBank accession nos. AY 303166, AY303167, and AY237157). Oligomers of23 nt that were completely complementary to the target sequence weresynthesized in vitro as a 2′-O-methyl oligoribonucleotides which had 52%GC content. Three versions of the probes were labeled with a linker atdifferent positions (between bases 8 and 9, 12 and 13, and 13 and 14 ofthe 23-mer) and an AE label attached at the linker by using methodspreviously described in detail (U.S. Pat. Nos. 5,185,439, 5,283,174, and5,656,744). The labeled probes (0.1 μmol per reaction) were individuallyhybridized at 60.deg.C. for 1 hr to a complementary synthetic ssRNAtarget sequence (10 μmol per reaction) in a 30 μl reaction mixturecontaining 15 μl of a hybridization reagent (190 mM succinate, 17% (w/v)lithium lauryl sulfate (LLS), 100 mM LiOH, 3 mM EDTA, and 3 mM EGTA, atpH 5.1). Then the hybridization mixture was diluted to 500 μl with thehybridization reagent and 20 μl aliquots were removed for performing thedetection step by adding to each detection reaction mixture 80 μl of thehybridization reagent, and then 200 μl of a selection reagent (600 mMboric acid, 182.5 mM NaOH, 1% (v/v) octoxynol (TRITON® X-100), at pH8.5), and hydrolysis was performed at 50.deg.C. for varying periods oftime. Then, production and detection of the signal was performed byadding 200 μl of a detection reagent (1 mM nitric acid and 32 mMH.sub.20.sub.2) followed by adding 200 μl of 1.5 M NaOH and thechemiluminescent signal (RLU) was measured (for 2 sec) by using aluminometer. The same detection reaction method was performed onaliquots that contained the individual probes without the ssRNA targetto measure hydrolysis of the AE label on unbound probes. From theseresults, the time at which half of the detectable label for each probecomposition was hydrolyzed (T.sub.½), when the complementary targetstrand was present or absent, was determined. The T.sub.½ for all threeprobes without the target (i.e., unbound probes) was between 0.69 and1.05 min, whereas when the target was present (i.e., bound probes) theT.sub.½ was between 25.8 and 125 min, indicating that the detectionprobes bound to the target and produced a detectable signal in excess ofthe background signal in a homogeneous reaction mixture. When probeswere hybridized with the target, they exhibited different T.sub.½characteristics: the probe labeled between positions 12 and 13 had theshortest T.sub.½ (25.8 min), the probe labeled between positions 13 and14 had the longest T.sub.½ (125 min), and the probe labeled betweenpositions 8 and 9 was an intermediate T.sub.½ (69 min). These resultsshow that all three probes were capable of binding to the complementaryRNA target, that labels in unbound probes could be distinguished fromlabels in bound probes by differential hydrolysis characteristics, andthat the labeling position on the oligomer affected the rate of labelhydrolysis so that optimal probes for an assay may be selected anddesigned using routine testing.

EXAMPLE 2 Sensitivity of Detection of Single-stranded andDouble-stranded Targets

The sensitivity of target detection was determined by using the sametarget and detection probe sequences as in Example 1, but comparingdetection of the RNA target when it was in ssRNA or dsRNA form. ThessRNA target oligomer and detection probe oligomer labeled with AEbetween positions 13 and 14 were used as in Example 1. In these assays,all reactions contained 0.1 μmol of the AE-labeled probe which washybridized to the ssRNA target present in a range of 0 to 5 fmol perhybridization reaction (100 μl hybridization mixtures incubated at60.deg.C. for 1 hr). Following hybridization, the AE label on unboundprobe was hydrolyzed by adding 200 μl of the selection reagent andincubating at 50.deg.C. for 10 min, and then the chemiluminescent signalfrom bound probe was detected substantially as described in Example 1.Results shown in Table 1, columns 1 and 2, demonstrate that a lineardetectable signal was measured over the range of target amounts tested.As little as 0.005 fmol of the ssRNA target in the reaction resulted ina detectable signal over the background signal obtained when no targetnucleic acid was present in the assay. “Net RLU” data (column 2) wascalculated by subtracting the background RLU (560 RLU when no target waspresent) from the detected RLU for each test sample.

In the tests performed using the dsRNA target, the target was made bysynthesizing a complementary RNA strand to the ssRNA target oligomerdescribed above and hybridizing the two complementary RNA strandstogether. The dsRNA target was tested substantially as described aboveby using the same probe as described above synthesized as a 2′-O-methyloligoribonucleotide and labeled with AE between positions 13 and 14.This detection probe was complementary to one of the strands in thedsRNA target. Before hybridization with the AE-labeled probe, the dsRNAtarget was denatured by heating it in solution (10 mM Li-succinate and0.01% LLS, pH5.0) at 90.deg.C. for 5-7 min, followed by quickly coolingon ice. In Test 1, 50 μmol of the target dsRNA was denatured and thendiluted to make the different amounts of target used in each of the 100μl hybridization reactions. In Test 2, the appropriate amounts of thetarget dsRNA were distributed to separate tubes in 50 μl aliquots, heatdenatured as described above, and then 50 μl of the hybridizationreagent containing the labeled probe was added to each tube make thehybridization reaction mixtures. The hybridization and detectionreactions were performed substantially as described above for the ssRNAtarget reactions and the results for the dsRNA target are shown in Table1, columns 3 to 5. The background signal detected when no target waspresent (942 RLU in Test 1, 932 RLU in Test 2) was subtracted from thedetected signal when the dsRNA target was present to obtain the “NetRLU” (column 4 for Test 1, and column 5 for Test 2). The Net RLUmeasurements showed that the assay produced a detectable signal that wasa substantially linear response over the range of target amounts tested.A positive signal was detected when as little as 0.01 fmol (Test 1) to0.05 fmol (Test 2) of the target RNA was present in the reactionindicating the sensitivity of the detection step.

TABLE 1 Signal Measured for Hybridization Reactions Containing DifferentAmounts of Target Net RLU- Net RLU- ssRNA (fmol) Net RLU dsRNA (fmol)Test 1 Test 2 0.005 391 — — — 0.01 719 0.01 122 — 0.02 1,289 0.02 151 —0.05 3,376 0.05 625 2,796 0.07 4,648 0.07 567 — 0.1 7,157 0.1 1,0665,561 0.25 16,922 0.25 2,079 — 0.5 29,729 0.5 4,443 27,142 1.0 64,9291.0 7,974 — 5.0 287,821 5.0 54,077 240,787

EXAMPLE 3 Capture and Detection of a Target RNA

In these assays, a capture probe capable of forming a hairpin structureunder hybridization conditions was used to capture a target nucleic acidfrom a sample, followed by hybridization of the target nucleic acid witha labeled detection probe and detection of a signal from bound detectionprobe. The capture probes used in these experiments all containstructural features that allow formation of a hairpin structure: a 5′region homopolymeric sequence, a 3′ region sequence that was fully orpartially complementary to the 5′ region sequence, and atarget-complementary sequence flanked by the 5′ and 3′ region sequences.The 5′ and 3′ region sequences form the “stem” portion of the hairpinstructure, and the target-complementary sequence forms the “loop”portion of the hairpin structure.

Three versions of a hairpin capture probe were synthesized and assayedusing routine methods to determine the Tm of the stem of the hairpincapture probe. The complementary 5′ and 3′ region sequences of all threeprobes were synthesized with deoxyribonucleotide linkages. Thetarget-complementary sequence of each of the hairpin probes was the23-nt target-complementary sequence as in Example 1, synthesized inprobes 1 and 2 with 2′-O-methyl linkages and in probe 3 withdeoxyribonucleotide linkages. In probes 1 and 3, the 5′ region was a(dT).sub.12 sequence and the 3′ region was a (dA).sub.12 sequence; andin probe 2, the 5′ region sequence was (dT).sub.5A(dT).sub.6 which ispartially complementary to the 3′ region sequence of (dA).sub.12.Schematically, the resulting capture probe sequences were as shown inlinear form below:

(probes 1 and 3; SEQ ID NO: 1) 5′TTTTTTTTTTTT - N.sub.23 - AAAAAAAAAAAA 3′, and (probe 2; SEQ ID NO: 2)5′ TTTTTATTTTTT - N.sub.23 - AAAAAAAAAAAA 3′,in which N.sub.23 represents the target-complementary sequence. In willreadily be appreciated that these linear sequences form partiallydouble-stranded hairpin structures by intramolecular hybridization ofthe 5′ region to the 3′ region and the target-complementary sequence(N.sub.23) becomes the loop portion of the hairpin structure. The Tm'sfor the double-stranded stem portions of these hairpin probes were in arange of about 46.deg.C. to about 57.deg.C. (46.3.deg.C. for probe 2,55.7.deg.C. for probe 3, and 56.7.deg.C. for probe 1).

Capture using these hairpin capture probes and detection of the targetwas performed using the dsRNA target and AE-labeled probe described inExample 2, using target amounts ranging from 0.05 to 5 fmol perreaction. To provide a sample similar to a clinical sample, the dsRNAtarget present in 200 μl of sample transport solution (110 mM LLS, 15 mMsodium phosphate monobasic, 15 mM sodium phosphate dibasic, 1 mM EDTA, 1mM EGTA, pH 6.7) was mixed with 200 μl of urine, to make a final samplevolume of 400 μl. This mixture was heated to denature the dsRNA target(at 90.deg.C. for 5 min, then cooled on ice), to provide a ssRNA targetstrand for hybridization with the capture probes. For each of thehairpin capture probes tested individually, the denatured RNA targetsample was mixed with 100 μl of a target capture reagent (250 mM HEPES,310 mM LiOH, 1.88 M LiCl, 100 mM EDTA, pH 6.4) containing 0.3 μmol ofthe hairpin capture probe and 50 μg of magnetic particles which were thecapture support (1 micron SERA-MAG.SUP.TM MG-CM particles, Seradyn, Inc.Indianapolis, Ind.), to which immobilized probe oligomers of dT.sub.14were covalently attached. The mixture was incubated at 65.deg.C. for 60min (a temperature above the Tm of each of the capture probes) and thenat room temperature for 30 min to form capture hybrids attached to theparticles. Then, the particles with the attached capture hybrids wereseparated magnetically from the liquid sample components which wereremoved. The particles with attached capture hybrids were washed twiceat room temperature with 500 μl of the sample transport solution andthen the particles with attached capture hybrids were separated from thesolution which was removed. The washed particles with the attachedcapture hybrids were mixed with 100 μl of water and heated (90.deg.C.for 5 min) to release the nucleic acid components of the capture hybrids(target and capture probe oligomers released into solution and theimmobilized probe remained covalently attached to the capture supportparticle). For detection of the target, the solution then was mixed withan AE-labeled detection probe, as described in Example 2, in 100 μl ofthe hybridization reagent and the mixture was incubated underhybridization conditions (55.deg.C. for 60 min) to allow the detectionprobe to bind to the target strands. Under these conditions, thereleased capture probes may reform the partially double-stranded hairpinstructure by intramolecular hybridization to minimize competitiveinhibition caused by the capture probes competing with the detectionprobes for hybridization to the target strand. The detection probe andthe target-complementary sequence of the hairpin capture probes will nothybridize to each other because they are the same sense strands.Detection of the signal from bound detection probes was performedsubstantially as described in Example 1, Control reactions withouttarget were treated identically and the background signal for allreactions was in the range of 535 to 715 RLU. The experimental resultsof these assays are shown in Table 2, column 2, as net RLU (detected RLUminus background RLU). For each assay, the ratio of the detected signalto background RLU is shown in Table 2, column 3.

TABLE 2 Assays Performed Using a Hairpin Capture Probe and DetectionProbe Target Amount (fmol) Net RLU Signal/Background Ratio 0.05 1,248 30.1 2,223 4.4 0.15 3,318 6.6 0.2 5,418 9 0.5 11,156 22 1.0 24,758 36 2.038,351 55 5.0 98,180 140

The results of these assays show that the combination of capture of atarget nucleic acid by using a hairpin capture probe and detection byusing a detection probe complementary to one strand of a dsRNA targeteffectively detected the target present in a sample for all of theamounts of target tested.

EXAMPLE 4 Assays Comparing Different Forms of Capture Probes

These assays compared the relative efficiency of capture and detectionof a target sequence, using methods similar to those described inExample 3, when the target capture step was performed by using a captureprobe of either a hairpin structure or linear structure. Unlessotherwise stated, the reagents used in these tests were the same asdisclosed in Examples 1 to 3 above. All of the assays used test samplescontaining 1 fmol of the ssRNA target, as described in Example 2, in 200μl of urine mixed with 200 μl of sample transport solution. For thetarget capture step, each 400 μl test sample was mixed with 100 μl oftarget capture reagent containing different amounts (0.1, 0.5, 1.0, 2.0,5.0, 10 and 20 pmoles) of either a partially double-stranded hairpincapture probe as described in Example 3 (SEQ ID NO:1) or a linearsingle-stranded capture probe of the following structure:

(SEQ ID NO: 3) 5′ X N.sub.23 TTTAAAAAAAAAAAA 3′that has substantially the same target-complementary sequence (N₂₃) asin the hairpin capture probe, but includes one additional 5′ nucleotide(X). In both the hairpin and linear forms of the capture probes, thetarget-complementary regions were synthesized with 2′-O-methyl linkages.In the hairpin capture probe, the target-complementary region wasflanked by the complementary 5′ poly-dT and 3′ poly-dA regions, whereasin the linear form the target-complementary region was covalently linkedto a 3′ (dT).sub.3(dA).sub.30 sequence. The reaction mixtures wereincubated at 65.deg.C. for 60 min and then at room temperature for 30min to allow formation of capture hybrids and attachment to the capturesupport via the immobilized probe. The supports with attached capturehybrids were separated from the liquid sample components by applying amagnetic field and then the supports were washed twice (using 0.5 ml ofsample transport solution each) substantially as is described in Example3. The final wash solution was removed and the capture supports withattached capture hybrids were mixed with 100 μl of water per assay,incubated at 90.deg.C. for 5 min and rapidly cooled on ice to releasethe capture hybrids into the nucleic acid components beforehybridization of the target strand with the detection probe. Then, eachtest sample was mixed with 0.1 pmole of the AE-labeled detection probeof Example 2 in 100 μl of hybridization reagent and incubated at55.deg.C. for 60 min to allow hybridization of the detection probe tothe target strand. Detection of the chemiluminescent signal fromdetection probes bound to the target strands was performed substantiallyas described in Example 3 (add 200 μl of selection reagent, incubate at55.deg.C. for 10 min, mix with 200 μl of detection reagent and measurechemiluminescence (for 5 sec) on a luminometer). The results are shownin Table 3, as net RLU in column 2 and 3, and the relative percentage ofdetection obtained when the capture step had been performed by using thelinear or hairpin forms of the capture probes, in columns 4 and 5. Thenet RLU was calculated by subtracting the background RLU from the RLUdetected in positive samples (background was 762 RLU for the hairpinprobe tests and 749 RLU for the linear probe tests). The relativepercentage of detection was calculated by setting the highest detectednet RLU at 100% (results for 0.1 pmole of hairpin capture probe) anddividing the lesser net RLU detected in the other tests by the highestnet RLU (21,778).

TABLE 3 Comparison of Hairpin and Linear Capture Probes Capture Net RLU% Detection Probe Hairpin Net RLU Hairpin % Detection (pmole) ProbeLinear Probe Probe Linear Probe 0.1 21,778 15,849 100 72.8 0.5 19,6147,286 90.1 33.5 1.0 18,248 4,172 83.8 19.2 2.0 17,316 2,578 79.5 11.85.0 11,889 1,137 54.6 5.2 10 9,778 722 44.9 3.3 20 4,519 458 20.7 2.1

These results show that the assays performed by using a linear form anda hairpin form of the capture probes specific for the same targetsequence resulted in a detectable signal for all of the assaysperformed. The relative percentage of detection was consistently higherwhen the capture probe was in the hairpin form compared to the linearform. The difference in relative percentage of detection ranged fromabout 10-fold more when the results obtained for the two forms werecompared for the highest amounts of capture probes tested (20 pmoles perreaction), to about 2.7-fold when the results obtained for the two formswere compared for the lowest amount of capture probes tested (0.1 pmoleper reaction). The differences between the assays that used the hairpinand linear capture probe forms may result from more competitiveinhibition when the linear capture probe was used because the releasedlinear form may compete with the detection probe for hybridization tothe target sequence during the detection phase of the assay whereas thehairpin form under the same conditions may reform the hairpin structureto limit competition between the target-complementary region of thecapture probe and the detection probe for binding the target.

EXAMPLE 5 Assays Using a Partially Double-Stranded Capture Probe

This example describes an embodiment that uses a completely or partiallydouble-stranded capture probe. The capture probe of this embodiment ismade up of two completely or partially complementary strands of whichone strand includes a target-complementary region that binds to aportion of the target nucleic acid. One version of the capture probe ismade up of a first capture probe strand (SEQ ID NO:4) and a secondcapture probe strand (SEQ ID NO:5) that are synthesized and hybridizedtogether to make a partially double-stranded capture probe bound byhybridization of at least their complementary 3′ polyA and 5′ polyTsequences as shown below.

(SEQ ID NO: 4) 5′TTTTTTTTTTTTTTTAGAGGATGGGTTTTCTAGGGG 3′ (SEQ ID NO: 5)3′AAAAAAAAAAAAAAATCTCTCTCTCTCTCTCTCTC 5′The oligomer of SEQ ID NO:4 contains a 5′ poly-T region and a 3′sequence complementary to a sequence contained in a human B19 parvovirusgenome (GenBank accession no. AY386330); and the oligomer of SEQ ID NO:5contains a 5′ poly-(TC) region and a 3′ poly-A region. In anotherversion, the capture probe is completely double stranded and made up ofthe first capture probe strand (SEQ ID NO:4) hybridized to itscomplementary strand (SEQ ID NO:6) as shown below.

(SEQ ID NO: 4) 5′TTTTTTTTTTTTTTTAGAGGATGGGTTTTCTAGGGG 3′ (SEQ ID NO: 6)3′AAAAAAAAAAAAAAATCTCCTACCCAAAAGATCCCC 5′

In separate assays, about 3.5 pmole of each version of the captureprobes is mixed with a 0.5 ml plasma sample containing parvovirus B19genomic DNA (denatured and, optionally, sheared or enzymaticallydigested into fragments of about 100 to 1000 nt long) and an equalvolume of target capture reagent containing capture support particleswith attached poly(A) oligomers as the immobilized probe. That is, theimmobilized poly(A) oligomers are complementary to the 5′ poly-dTportion of the capture probe oligomer of SEQ ID NO:4. The mixture isincubated (60-65.deg.C., 20-60 min) to allow the capture probes todissociate into the component oligomers (SEQ ID NO:4 and SEQ ID NO:5, orSEQ ID NO:4 and SEQ ID NO:6), to allow the target-specific portion ofSEQ ID NO:4 to hybridize to the complementary sequence in the parvovirusB19 target DNA. Then, the mixture is incubated at a lower temperature(25-30.deg.C., 14-30 min) to allow the complementary homopolymericsequences of the capture probe and the immobilized probe to hybridize,thereby attaching the target B19 DNA to the magnetic particles in acapture hybrid that includes the capture probe strands of SEQ ID NO:5hybridized to the complementary sequence in the parvovirus B19 DNA.Particles with the attached capture hybrids are separated from thesample components by applying a magnetic force to the outside of thecontainer and the liquid sample components, including unhybridizedcapture probe strands, are removed. A washing step is used in which theparticles with the attached capture hybrids are suspended in an aqueoussolution of sufficient ionic strength the maintain the capture hybridattached to the capture support, then the particles are separated fromthe aqueous solution using magnetic force, and the washing solution withany unbound capture probe oligomers and other sample components isremoved. For detection of the captured B19 DNA, the particles with theattached capture hybrids are mixed with a hybridization reagentcontaining detection probe oligomers of SEQ ID NO:7 (0.1-0.5 pmoles perreaction) labeled with a fluorescent label (e.g., fluorescein) and thedetection probes are hybridized to a complementary sequence in thecaptured B19 DNA by incubating the mixture below the Tm of the detectionprobe but at a temperature above the melting temperature of thepolA-polyT duplex, to release the B19 target nucleic acid into thesolution phase (e.g., 55.deg.C. for 20-60 min). The mixture is thenincubated at a lower temperature (e.g., 25-30.deg.C. for 10-30 min) toallow hybridization complexes made up of the B19 target DNA, detectionprobe and the poly-dT containing capture probe strand to attached to thepoly (A) immobilized probes on the particles. The particles withattached complexes that contain the hybridized detection probes areseparated from the solution phase as described above and the solutionphase containing unbound detection probes is discarded. The particleswith attached complexes are optionally washed, substantially asdescribed above, under conditions that maintain the hybridizationcomplexes on the particles to remove remaining unbound detection probes.Finally, the particles are mixed with a volume of liquid (e.g., 0.5 mlwater) and the fluorescence of the mixture is measured using standardfluorometric procedures. A negative control sample, i.e. plasmacontaining no B19 particles or DNA, is treated identically as describedabove and the fluorescence that is measured from the negative controlsample indicates background signal for the assay. For both the partiallyand completely double-stranded versions of the capture probe, theseassays produce a detectable positive signal for test samples thatcontain parvovirus B19 nucleic acid that is significantly greater thanthe background signal that negative control samples produce. Positivesignals indicate the presence of the parvovirus B19 target nucleic acidin the samples.

The invention claimed is:
 1. A kit for the detection of a target nucleicacid, wherein the kit comprises: a capture probe having the structure5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Y each comprise nucleicacid sequences that can form a double stranded portion of a hairpinstructure and wherein one of X or Y is a capture sequence furthercomprising a first member of a specific binding pair and the other of Xor Y is a terminal sequence of the hairpin capture probe, and wherein a′b′ c′ comprises a target-complementary sequence that hybridizesspecifically to a target sequence in the target nucleic acid and isflanked by X and Y to thereby form a loop portion of the hairpinstructure; and a detection probe that hybridizes specifically to atarget sequence that overlaps or is the same as the target sequencehybridized by the target-complementary sequence of the capture probe. 2.The kit of claim 1, wherein the detection probe hybridizes specificallyto the same target sequence hybridized by the target-complementarysequence of the capture probe.
 3. The kit of claim 1, wherein thedetection probe comprises a target-complementary sequence that is a′ b′c′.
 4. The kit of claim 1 that further includes an immobilized probeattached to a capture support, wherein the immobilized probe includes asecond member of a specific binding pair that binds specifically to thecapture probe's first member of a specific binding pair.
 5. The kit ofclaim 4, wherein the first and second specific binding pair members aresubstantially complementary sequences that hybridize the capture probeto the immobilized probe under hybridizing conditions.
 6. The kit ofclaim 5, wherein the first member of the specific binding pair is asequence from 10 to 30 nucleobases in length and is substantially apoly-A sequence.
 7. The kit of claim 5, wherein the second member of thespecific binding pair is a sequence from 10 to 30 nucleobases in lengthand is substantially a poly-A sequence.
 8. The kit of claim 1, whereinsaid target complementary sequence is synthesized with 2′-O-methyllinkages.
 9. The kit of claim 1, wherein Y is the capture sequence thatforms the first member of the specific binding pair.
 10. The kit ofclaim 9, wherein the capture probe has the structure 5′-X.sub.n a′b′c′poly(A).sub.10-30.
 11. A reaction mixture for the detection of a targetnucleic acid, wherein the reaction mixture comprises: a capture probehaving the structure 5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Yeach comprise nucleic acid sequences that can form a double strandedportion of a hairpin structure and wherein one of X or Y is a capturesequence further comprising a first member of a specific binding pairand the other of X or Y is a terminal sequence of the hairpin captureprobe, and wherein a′ b′ c′ comprises a target-complementary sequencethat hybridizes specifically to a target sequence in the target nucleicacid and is flanked by X and Y to thereby form a loop portion of thehairpin structure; and a detection probe that hybridizes specifically toa target sequence that overlaps or is the same as the target sequencehybridized by the target-complementary sequence of the capture probe.12. The reaction mixture of claim 11, wherein the detection probehybridizes specifically to the same target sequence hybridized by thetarget-complementary sequence of the capture probe.
 13. The reactionmixture of claim 11, wherein the detection probe comprises atarget-complementary sequence that is a′ b′ c′.
 14. The reaction mixtureof claim 11, wherein the first member of the specific binding pair is asequence from 10 to 30 nucleobases in length and is substantially apoly-A sequence.
 15. The reaction mixture of claim 11, wherein thesecond member of the specific binding pair is a sequence from 10 to 30nucleobases in length and is substantially a poly-A sequence.
 16. Thereaction mixture of claim 11, wherein said target complementary sequenceis synthesized with 2′-O-methyl linkages.
 17. The reaction mixture ofclaim 11, wherein Y is the capture sequence that forms the first memberof the specific binding pair.
 18. The reaction mixture of claim 17,wherein the capture probe has the structure 5′-X.sub.n a′b′c′poly(A).sub.10-30.
 19. The reaction mixture of claim 11, wherein thedetection probe is labeled with a chemiluminescent compound.
 20. Thereaction mixture of claim 19, wherein the chemiluminescent compound isan acridinium ester.
 21. The reaction mixture of claim 11, wherein thedetection probe is labeled with a fluorescent compound.
 22. The reactionmixture of claim 21, wherein the detection probe is a molecular torch ora molecular beacon.
 23. The reaction mixture of claim 11, wherein thereaction mixture contains the target nucleic acid, and wherein thedetection probe is hybridized to the target nucleic acid.